Simian spumaretrovirus (simian foamy viruses, SFV) have been identified as potentially confounding agents for AIDS research utilizing macaques and their tissues. In addition to lost experiments due to the lytic effects of SFV in primary macaque cell cultures, there is evidence suggesting that persistent, non-lytic SFV infection may influence replication of lentiviruses. Further, recent studies of transgenic mice harboring human SFV genes suggest that spumaretroviruses may, in fact, be pathogenic. We and others have shown that the polymerase chain reaction (PCR) is less sensitive than virus isolation for detecting SFV in macaque tissues, probably because of the lower number of cells that can be used in the PCR assay. Accordingly, we explored the sensitivity of established cell lines and primary rhesus macaque fetal cells for isolating SFV from macaque peripheral blood leukocytes (PBLs). Primary mixed mesenchymal cell cultures derived from 70 days gestational aged rhesus macaques co-cultivated with PBLs for 21 days identified approximately 25% more SFV-infected macaques than similar assays using established cell lines. We have now used this assay system to estimate the incidence of SFV infection in Celebes, rhesus and Japanese macaques 28% (7 of 25) Celebes macaques, 50.5% (93 of 184) rhesus macaques and 18.9% (43 of 228) Japanese macaques yielded SFV isolates. The incidence was age-influenced in rhesus macaques with a 15.4% incidence in animals less than 1 year of age. Thus, a substantial number of primary macaque cell cultures can be expected to contain SFV unless measures are taken to exclude this agent from macaque resources.